HIGH THROUGH-PUT AUTOMATED DNA SEQUENCING

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We request funding for instrumentation to update a successful central DNA sequencing core facility at the California Institute of Technology. The Core Facility has developed over the last five years into an indispensable contributor to the ongoing research projects of Biology, Chemistry, Geology, and Environmental Engineering Divisions. Currently we are producing about 2500 sequences a month for walk-in users. In addition, the Core Facility has carried out two production scale sequencing projects coordinated by the Genome Resource Center at Caltech, completing the sequence of an entire micro-organism, Pyrobaculum aerophilum, and a large region of human chromosome 16, in addition to many BAC clones. All labs at Caltech now rely on automated, fluorescent-based sequencing. In order to continue the quality of service we have achieved in the face of increasing demand, we are requesting three types of equipment: an ABI Prism 3700 DNA Analysis system, new PCR machines, and several small computers. We have chosen the ABI Prism 3700, which is based on a novel capillary electrophoresis system rather than slab gels, because of its promise of the highest through-put, with the least labor at the lowest cost and with the highest quality of any such instrument currently available. The PCR machines will be needed to exploit the capacity of the ABI3700. The computers are needed to interface the new equipment with the existing Sequence Analysis Facility which distributes the sequence to the users. Twenty-eight major users provide abstracts of the work they will do. There will continue to be standard sequencing of single cDNAs, PCR products, site-directed mutants, etc. This will provide service to projects covering, among others, genes involved in RNA splicing, molecular control of neural crest and/or muscle development, in controlling body segment development in Drosophila, in cell cycle regulation in Xenopus and yeast, in cell fate and morphogenesis in several organisms, genes involved in floral development in Arabidopsis. Availability of the enormous capacity of the 3700 will, we hope, also drive new approaches in the laboratories here. For instance, several labs (Anderson, Wold, Meyerowitz) are developing strategies to identify cDNA markers based on differential gene expression for various specific cell types and individual stages of development. Another project proposes to screen age-related mutations in human mitochondria. Another dimension is sequencing entire microorganisms and panning for organisms with interesting known pathways. These latter projects could involve many thousands of runs per month in themselves. Thus, we hope to maintain our ability to function both as a walk-in facility and a high-tech production scale facility, as we have done in the past.